Abstract
The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of approximately 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, tumor rejection antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers.
Highlights
From the Departments of ‡Biochemistry and §Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 753909038, the **Tissue Proteomics Unit, Center for Biologics, Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20857, and the ¶Pathogenetics Unit, Laboratory of Pathology and Urologic Oncology Branch, the Laboratory of Pathology and the ʈCancer Prevention Studies Branch, NCI, National Institutes of Health, Bethesda, Maryland 20857
The differential in-gel electrophoresis (DIGE) technique recently introduced by Amersham Biosciences, Inc. is aimed at improving reproducibility
The upper panel shows the images of the protein spots in the 2D gel, and the lower panel shows the 3D image of the corresponding spots and their calculated spot volumes
Summary
2D, two dimensional; CHAPS, 3-([(3cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Cy3, 1-(5-carboxypentyl)-1Ј-propylindocarbocyanine halide N-hydroxysuccinimidyl ester; Cy5, 1-(5-carboxypentyl)-1Ј-methylindodi-carbocyanine halide N-hydroxysuccinimidyl ester; DIGE, differential in-gel electrophoresis; HPLC, high performance liquid chromatography; MS, mass spectrometry; LCQ, liquid chromatography electrospray ion trap mass spectrometer; LCM, laser capture microdissection; DTT, dithiothreitol; 3D, three dimensional. Quantitation of the protein profile can be rapidly and accurately achieved based on the fluorescence intensity This technique was used by Davison and colleagues [18] for proteomics studies of mouse liver homogenates to examine the molecular basis of the hepatotoxin, N-acetyl-p-aminophenol. We have applied the DIGE technique for the identification of esophageal squamous cell cancer-specific protein markers Both esophageal cancer and normal squamous epithelium cells were procured from the same esophageal tumor sample by laser capture microdissection (LCM). Three protein spots were identified by mass spectrometry and validated by Western blotting analysis To our knowledge, this is the first global quantitation of differential protein expression analysis by 2D-PAGE between LCM cancer cells and normal cells from the same human tumor-tissue sample
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